Nucleic acid polymer chemistry has played a crucial role in many developing technologies in the pharmaceutical, diagnostic, and analytical fields, and more particularly in the subfields of antisense and anti-gene therapeutics, combinatorial chemistry, branched DNA signal amplification, and array-based DNA diagnostics and analysis (e.g., Uhlmann and Peyman, Chemical Reviews, 90:543-584, 1990; Milligan et al., J. Med. Chem. 36:1923-1937, 1993; DeMesmaeker et al., Current Opinion in Structural Biology, 5:343-355, 1995; Roush, Science, 276:1192-1193, 1997; Thuong et al., Angew. Chem. Int. Ed. Engl., 32:666-690, 1993; Brenner et al., Proc. Natl. Acad. Sci., 89:5381-5383, 1992; Gold et al., Ann. Rev. Biochem., 64:763-797, 1995; Gallop et al., J. Med. Chem., 37:1233-1258, 1994; Gordon et al., J. Med. Chem., 37:1385-1401, 1994; Gryaznov, International application No. PCT/US94/07557; Urdea et al., U.S. Pat. No. 5,124,246; Southern et al., Genomics, 13:1008-1017, 1992; McGall et al., U.S. Pat. No. 5,412,087; Fodor et al., U.S. Pat. No. 5,424,186; Pirrung et al., U.S. Pat. No. 5,405,783).
Much of this chemistry has been directed to improving the binding strength, specificity, and nuclease resistance of natural nucleic acid polymers, such as DNA. Unfortunately, improvements in one property, such as nuclease resistance, often involve trade-offs against other properties, such as binding strength. Examples of such trade-offs abound: peptide nucleic acids (PNAs) display good nuclease resistance and binding strength, but have reduced cellular uptake in test cultures (e.g., Hanvey et al., Science, 258:1481-1485, 1992); phosphorothioates display good nuclease resistance and solubility, but are typically synthesized as P-chiral mixtures and display several sequence-non-specific biological effects (e.g., Stein et al., Science, 261:1004-1012, 1993); methylphosphonates display good nuclease resistance and cellular uptake, but are also typically synthesized as P-chiral mixtures and have reduce duplex stability (e.g., DeMesmaeker et al. (cited above); and so on.
Recently, a new class of oligonucleotide analog has been developed having so-called N3′→P5′ phosphoramidate internucleoside linkages which display favorable binding properties, nuclease resistance, and solubility (Gryaznov and Letsinger, Nucleic Acids Research, 20:3403-3409, 1992; Chen et al., Nucleic Acids Research, 23:2661-2668, 1995; Gryaznov et al., Proc. Natl. Acad. Sci., 92:5798-5802, 1995; and Gryaznov et al., J. Am. Chem. Soc., 116:3143-3144, 1994). Phosphoramidate compounds contain a 3′-amino group at each of the 2′-deoxyfuranose nucleoside residues replacing a 3′-oxygen atom. The synthesis and properties of oligonucleotide N3′→P5′ phosphoramidates are also described in Gryaznov et al., U.S. Pat. Nos. 5,591,607; 5,599,922; 5,726,297; and Hirschbein et al., U.S. Pat. No. 5,824,793.
Oligonucleotides with various modifications of the internucleoside linkages and 2′-position of the sugar rings have been described. Among these compounds are phosphodiester (PO), and phosphorothioate (PS) oligonucleotides containing 2′-fluoro substituents in ribo- or in arabino-configurations (Kawasaki et al., J. Med. Chem. 36:831-841, 1993; Ikeda et al., Nucl. Acids Res., 26:2217-2244, 1998; Kois et al., Nucleosides Nucleotides, 12:1093-1109, 1993). Of these the oligo-2′-ribo-fluoronucleotides form the most stable complexes with DNA and RNA, whereas stability of duplexes formed by the isomeric oligo-2′-arabino-fluoro nucleotides is significantly lower. The duplex stabilizing effects of 2′-ribo-fluoronucleotides was mainly attributed to their C3′-endo or N-type sugar puckering (Kawasaki et al., J. Med. Chem. 36:831-841, 1993). Unfortunately, phosphodiester oligo-2′-ribo-fluoronucleotides are not resistant to hydrolysis by cellular nucleases, and require further modification of the internucleoside linking groups for any in vivo applications of these compounds. Therefore, more stable oligonucleotide phosphorothioate (Kawasaki et al., J. Med. Chem. 36:831-841, 1993) and N3′→P5′ phosphoramidates (Schultz et al., Nucl. Acids Res., 24:2966-2973, 1996), which resist enzymatic digestion were prepared. For the former compounds introduction of phosphorothioate linkages resulted in reduction of the duplex stability, whereas for the latter ones synergistic stabilizing effects of both 2′-ribo-fluoro and 3′-amino groups were observed. Additionally, oligo-2′-ribo-fluoro-N3′→P5′ phosphoramidates were less acid labile than their 2′-deoxy N3′→P5′ phosphoramidate counterparts (Schultz et al., Nucl. Acids Res., 24:2966-2973, 1996).
The oligonucleotide N3′→P5′ phosphoramidates form unusually stable duplexes with complementary DNA and especially RNA strands, as well as stable triplexes with DNA duplexes, and they are also resistant to nucleases (Chen et al., Nucleic Acids Research, 23:2661-2668, 1995; Gryaznov et al., Proc. Natl. Acad. Sci., 92:5798-5802 1995). Moreover oligonucleotide N3′→P5′ phosphoramidates were found to be more potent antisense agents than phosphorothioate derivatives both in vitro and in vivo (Skorski et al., Proc. Natl. Acad. Sci., 94:3966-3971, 1997). At the same time the phosphoramidates apparently have a low affinity to the intra- and extracellular proteins and increased acid liability relative to the natural phosphodiester counterparts (Gryaznov et al., Nucleic Acids Research, 24:1508-1514, 1996). These two features of the oligonucleotide phosphoramidates may potentially adversely effect their pharmacological properties for some applications. In particular, the acid stability of an oligonucleotide is an important quality given the desire to use oligonucleotide agents as oral therapeutics.
In order to circumvent the above described problems associated with presently known oligonucleotide analogs, a new class of compounds was sought that embodies the best characteristics from both oligonucleotide phosphoramidates and 2′-ribo-fluoronucleotides. The present invention describes the synthesis, properties and uses of oligonucleotide analogues containing 2′-arabino-fluoronucleosides and internucleoside N3′→P5′ phosphoramidate linkages.